Sandra Sanabria

Inverse agonist histamine H3 receptor PET tracers labelled with carbon-11 or fluorine-18

Two histamine H3 receptor (H3R) inverse agonist PET tracers have been synthesized and characterized in preclinical studies. Each tracer has high affinity for the histamine H3 receptor, has suitable lipophilicity, and neither is a substrate for the P‐glycoprotein efflux pump. A common phenolic precursor was used to synthesize each tracer with high specific activity and radiochemical purity by an alkylation reaction using either [11C]MeI or [18F]FCD2Br. Autoradiographic studies in rhesus monkey and human brain slices showed that each tracer had a widespread distribution with high binding densities in frontal cortex, globus pallidus and striatum, and lower uptake in cerebellum. The specificity of this expression pattern was demonstrated by the blockade of the autoradiographic signal by either the H3R agonist R‐α‐methylhistamine or a histamine H3R inverse agonist. In vivo PET imaging studies in rhesus monkey showed rapid uptake of each tracer into the brain with the same distribution seen in the autoradiographic studies. Each tracer could be blocked by pretreatment with a histamine H3R inverse agonist giving a good specific signal. Comparison of the in vitro metabolism of each compound showed slower metabolism in human liver microsomes than in rhesus monkey liver microsomes, with each compound having a similar clearance rate in humans. The in vivo metabolism of 1b in rhesus monkey showed that at 60 min, ∼35% of the circulating counts were due to the parent. These tracers are very promising candidates as clinical PET tracers to both study the histamine H3R system and measure receptor occupancy of H3R therapeutic compounds. Synapse 63:1122–1132, 2009.

In vitro and in vivo properties of 3-tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d]-[1,2,4]triazine (MRK-016), a GABAA receptor alpha5 subtype-selective inverse agonist

3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human α1-, α2-, α3-, and α5-containing GABAA receptors. It has inverse agonist efficacy selective for the α5 subtype, and this α5 inverse agonism is greater than that of the prototypic α5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (α5IA). Consistent with its greater α5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than α5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC50 value of 15 ng/ml that was similar to the rhesus monkey plasma EC50 value of 21 ng/ml obtained using [11C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3–0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species (∼3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.

Benzodiazepine Binding Site Occupancy by the Novel GABAA Receptor Subtype-Selective Drug 7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) in Rats, Primates, and Humans

The GABAA receptor α2/α3 subtype-selective compound 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023; also known as MK-0777) is a triazolopyridazine that has similar, subnanomolar affinity for the benzodiazepine binding site of α1-, α2-, α3-, and α5-containing GABAA receptors and has partial agonist efficacy at the α2 and α3 but not the α1 or α5 subtypes. The purpose of the present study was to define the relationship between plasma TPA023 concentrations and benzodiazepine binding site occupancy across species measured using various methods. Thus, occupancy was measured using either in vivo [3H]flumazenil binding or [11C]flumazenil small-animal positron emission tomography (microPET) in rats, [123I]iomazenil γ-scintigraphy in rhesus monkeys, and [11C]flumazenil PET in baboons and humans. For each study, plasma-occupancy curves were derived, and the plasma concentration of TPA023 required to produce 50% occupancy (EC50) was calculated. The EC50 values for rats, rhesus monkeys, and baboons were all similar and ranged from 19 to 30 ng/ml, although in humans, the EC50 was slightly lower at 9 ng/ml. In humans, a single 2-mg dose of TPA023 produced in the region of 50 to 60% occupancy in the absence of overt sedative-like effects. Considering that nonselective full agonists are associated with sedation at occupancies of less than 30%, these data emphasize the relatively nonsedating nature of TPA023.

Gender and age affect NK1 receptors in the human brain - a positron emission tomography study with [18F]SPA-RQ.

Substance P (SP) is a neurotransmitter and neuromodulator that mediates its effects in the brain predominantly via the neurokinin-1 receptors (NK1Rs). NK1Rs and SP have been shown clinically to be involved in nausea and emesis after chemotherapy (CINV) and have been implicated preclinically in a range of neuropsychiatric disorders but unlike CINV their blockade in these conditions does not have proven clinical value. We investigated whether age and gender affects NK1R binding potential (NK1R-BP; an index of receptor availability) in the living human brain using PET and [18F]SPA-RQ, a highly specific NK1R antagonist. Forty-five healthy volunteers (35 male and 10 female), aged between 19 and 55 years were studied. NK1R-BP was estimated using the simplified reference tissue model with cerebellum as a reference region. A regression analysis indicated that that a loss of NK1R is associated with normal ageing as shown by decreased NK1R-BP (average rate 7% per decade). Statistically significant negative associations between age and NK1R-BP were observed in temporal, parietal and frontal cortex, hippocampus and parahippocampal formation. In addition preliminary data were obtained suggesting possible gender differences in NK1R-BP in the cortex and putamen with females having a lower NK1R-BP. The exact physiological significance of these results remains to be elucidated but conceptually they could be involved in age-related CNS disorders or those with gender differences in prevalence.

Efficient radiosynthesis of 3'-deoxy-3'-18F-fluorothymidine using electrowetting-on-dielectric digital microfluidic chip.

Access to diverse PET tracers for preclinical and clinical research remains a major obstacle to research in cancer and other disease research. The prohibitive cost and limited availability of tracers could be alleviated by microfluidic radiosynthesis technologies combined with a high-yield microscale radiosynthetic method. In this report, we demonstrate the multistep synthesis of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) with high yield on an electrowetting-on-dielectric (EWOD) microfluidic radiosynthesizer, previously developed in our group. We have identified and established several parameters that are most critical in the microscale radiosynthesis, such as the reaction time, reagent concentration, and molar ratios, to successfully synthesize 18F-FLT in this compact platform. Methods: 18F-FLT was synthesized from the 3-N-Boc-1-[5-O-(4,4′-dimethoxytrityl)-3-O-nosyl-2-deoxy-β-d-lyxofuranosyl] thymine precursor on the EWOD chip starting from the first solvent exchange and 18F-fluoride ion activation step to the final deprotection step. The fluorination reaction was performed in a mixture of thexyl alcohol and dimethyl sulfoxide. The crude product after deprotection was collected from the chip and purified on a custom-made solid-phase extraction cartridge and subjected to quality control testing. The purified 18F-FLT was suitable for small-animal PET studies in multiple nude mice xenografted with the A431 carcinoma cell line. Results: 18F-FLT was successfully synthesized on the EWOD microdevice coupled with an off-chip solid-phase extraction purification with a decayed-corrected radiochemical yield of 63% ± 5% (n = 5) and passed all of the quality control tests required by the U.S. Pharmacopeia for radiotracers to be injected into humans. We have successfully demonstrated the synthesis of several batches of 18F-FLT on EWOD, starting with approximately 333 MBq of radioactivity and obtained up to 52 MBq (non–decay-corrected) of 18F-FLT on cartridge purification. The specific activity of 2 representative preparations of 18F-FLT synthesized on the EWOD chip were measured to be 1,800 and 2,400 GBq/μmol. Conclusion: The EWOD microchip and optimized synthesis method in combination represent an effective platform for synthesizing 18F-FLT with high yield and of good quality for imaging. This compact platform, with configurable synthesis steps, could potentially form the basis of a stand-alone system that decouples PET probe production from the cyclotron and specialized radiochemistry facilities and increases diversity and flexibility in probe production.

Discovery of MK-3168: A PET Tracer for Imaging Brain Fatty Acid Amide Hydrolase.

We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.

PET imaging in healthy subjects and migraineurs suggests CGRP receptor antagonists do not have to act centrally to achieve clinical efficacy.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and sensory neuropeptide implicated in the pathophysiology of migraine headache. CGRP receptor (CGRP-R) antagonists, including telcagepant, have shown clinical efficacy in treating migraine. CGRP-Rs are expressed in the CNS, particularly in the brainstem and cerebellum as well as in the periphery on vascular smooth muscle cells. To investigate whether central CGRP-Rs were likely to be involved in the anti-migraine effects of CGRP-R antagonists we examined central CGRP-R occupancy (CGRP RO) at an efficacious dose of telcagepant in healthy volunteers and in migraineurs during ictal and interictal periods using the novel PET tracer [11C]MK-4232. CGRP RO was evaluated in healthy subjects (n=3) at the lowest clinically efficacious dose (140 mg, PO) of telcagepant ~2h after dosing, coinciding with the time point of efficacy evaluation in clinical migraine studies. PET imaging showed only low CGRP RO (4% - 10%) which is within test-retest variability, suggesting that central activity is not a pre-requisite for anti-migraine efficacy of CGRP-R antagonists. A supratherapeutic telcagepant dose (1120 mg, PO) produced only moderate (43-58%, n=4) CGRP RO at ~Tmax (3h after administration). Subsequently, the possibility that brain penetration of telcagepant, a P-gp brain efflux pump substrate, may be increased in migraine patients or during migraine by blood brain barrier opening, was investigated by PET studies in the ictal and interictal periods in migraineurs (n=3) ~2 h after telcagepant dosing (140 mg, PO). Comparison of [11C]MK-4232 PET study results from ictal and interictal periods in migraineurs to healthy volunteers, suggests similar low RO and no significant differences between states. In conclusion, PET studies with the CGRP-R PET tracer [11C]MK-4232 after therapeutic doses of telcagepant in healthy volunteers and migraineurs suggest that central antagonism of CGRP-R is not necessary for therapeutic efficacy in migraine pain relief and that migraine pain is therefore at least in part peripheral in origin.

Occupancy of human brain GABAA receptors by the novel α5 subtype-selective benzodiazepine site inverse agonist α5IA as measured using [11C]flumazenil PET imaging

GABA(A) receptor α5-selective inverse agonists enhance cognitive performance in pre-clinical species. However, a key aspect of the clinical development of such compounds is the demonstration that in man such compounds are devoid of the anxiogenic-like activity associated with non-selective inverse agonists such as FG 7142. The triazolophthalazine α5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is an α5-selective inverse agonist which enhances cognitive performance in rodents and encouragingly in human Phase I Safety and Tolerability studies it was devoid of the anxiogenic-like activity associated with FG 7142. However, in order to appropriately interpret this latter observation, it was considered important to demonstrate that the absence of anxiogenic-like activity occurs at significant levels of receptor occupancy. Consequently, the occupancy of human brain GABA(A) receptors was measured using [¹¹C]flumazenil positron emission tomography in three healthy normal young male volunteers following a single oral dose of 2 mg α5IA. One hour after dosing, mean occupancy levels were 53% and this fell to 16% by 8 h post-dose, with the plasma α5IA concentration corresponding to 50% occupancy being 10 ng/mL. These data clearly show that an α5-selective inverse agonist is not associated with anxiogenic-like side effects at doses that give ~50% occupancy.

Characterization of [18F]Y1-973, a novel PET tracer for the neuropeptide Y Y1 receptor (NPY Y1), in rhesus monkey

Introduction: NPY Y1 has been implicated in the regulation of energy balance in humans, and therefore NPY Y1 antagonists have been proposed as potential therapeutics for the treatment of obesity. A PET tracer for NPY Y1 would be useful to study the interaction of small molecule NPY Y1 antagonists with NPY Y1. We have radiolabeled a novel NPY Y1 antagonist with F for use in pre-clinical PET studies to evaluate NPY Y1 antagonists as potential drug candidates. We report here on the synthesis, characterization, and use of [F]Y1-973 for receptor occupancy studies with NPY Y1 antagonist Y1-718 in rhesus monkey.

In vitro Characterization of MK-3328: A Novel Fluorinated Positron Emission Tomography Tracer for Plaques

IC-P-098 IN VITRO CHARACTERIZATION OF MK-3328: A NOVEL FLUORINATED POSITRON EMISSION TOMOGRAPHY TRACER FOR BETA-AMYLOID PLAQUES Cyrille Sur, Zhizhen Zeng, Eric Hostetler, Brett M. Connolly, Patricia J. Miller, Stacey O’Malley, Tsin-Bau Chen, Christopher Culberson, Scott Harrison, Jim Mulhearn, Scott Wolkenberg, James Barrow, Sandra Sanabria, Jacquelynn J. Cook, Richard Hargreaves, David L. Williams, Imaging Merck and Co Inc., West Point, PA, USA; Medicinal Chemistry Merck and Co Inc., West Point, PA, USA; Neuroscience Franchise Merck and Co Inc, West Point, PA, USA. Contact e-mail: cyrille_sur@merck.com